DNA Methylation and RNA methylation are processes facilitated by methyltransferases, where a methyl group (CH3) is added to a specific atom on a DNA or RNA molecule. In cellular RNA, over 100 types of chemical modifications have been identified. Various RNA methylation modifications include m6A RNA methylation, m5C RNA methylation, m1A RNA methylation, m7G RNA methylation, and more. Among these, the most prominent and extensively studied is m6A RNA methylation, occurring at the sixth nitrogen atom of the adenine residue in RNA molecules (N6-methyladenosine, m6A). This modification represents the most common post-transcriptional modification in eukaryotic mRNA, constituting approximately 80% of all RNA methylation modifications.
MeRip-seq is based on the principle of antibody-specific binding to methylated bases. It utilizes m6A-specific antibodies for immunoprecipitation to enrich RNA fragments that undergo methylation. High-throughput sequencing is then employed to detect transcripts with m6A modifications. However, this method can only identify regions with high methylation and lacks single-base resolution in recognizing RNA methylation.
Nanopore sequencing is a long read sequencing technology based on electric signal recognition of base sequences. Different modifications on RNA bases cause variations in the obstruction size as they pass through a nanopore channel, generating characteristic electrical signals. Real-time monitoring of these signals allows for the determination of the corresponding base types and whether they carry base modifications. In other words, Nanopore sequencing can directly perform full-length transcriptome sequencing on natural RNA samples without the need for specific antibody binding. It enables the detection of m6A (N6-methyladenosine) methylation modifications on RNA with single-base resolution, while simultaneously providing quantitative gene/transcript expression and transcript structure identification.
Nanopore sequencing technology leverages the detection of signals generated when a single molecule passes through a nanopore, causing a potential difference on either side of the pore. The nanopore's diameter allows only individual nucleotide polymers to pass through. The charged nature of different bases, including those with methylated modifications, varies, enabling the detection of corresponding base types and methylation information through differences in electrical signals.
Single Nucleotide Resolution Across the Entire Transcriptome: Identification of m6A methylation modifications at single nucleotide resolution throughout the transcriptome.
Direct Reading of m6A Methylation Information: Direct extraction of m6A methylation information without the need for immunoprecipitation enrichment experiments.
Simultaneous Quantification of Gene/Transcript Expression and Transcript Structure: Conduction of both quantitative analysis of gene/transcript expression and investigation of transcript structure concurrently.
Absence of Reverse Transcription and PCR Bias: Nanopore direct RNA sequencing eliminates the need for disruption, reverse transcription, and PCR amplification, allowing for the direct full-length transcriptome sequencing of natural RNA samples.
(Nicky Jonkhout et al,. RNA 2017)
m6A RNA Methylation Detection:
Comparative analysis of m6A methylation levels across different motifs.
Comparative analysis of m6A methylation levels between different samples.
Functional investigation of m6A methylation levels.
Quantification of Gene/Transcript Expression and Identification of Transcript Structural Variations:
Accurate quantification of gene/transcript expression at the transcript level to identify differentially expressed transcripts.
Identification of functional genes.
Precise detection of variable splicing, alternative polyadenylation (APA), fusion genes, etc.
Correlation analysis between m6A RNA methylation and gene/transcript expression.